The laboratory uses advanced microscopy imaging to study cellular and molecular mechanisms that underlie mechano-transduction in auditory and vestibular sensory organs. Three unconventional myosins: myosins VI, VIIA and XV have a vital role in hair cell stereocilia formation and function because they are specifically expressed in or around the stereocilia and mutations in their genes lead to stereocilia malformation and deafness. We have been using a combination of microscopy and immunocytochemical techniques to examine the precise localization of these proteins within the structure of the stereocilia. We have also been using the yeast two-hybrid assay to screen a mouse Organ of Corti cDNA library for potential protein partners directly involved in stereocilia development and function. To date several promising clones have been identified to specific regions of Myosin XV and Espin, another actin binding protein essential for stereocilia function. Experiments over the past year have also focused on determining whether prestin, a recently cloned OHC plasma membrane protein postulated to be responsible for electromotility, is present in hair cells of the vestibular system. Using RT-PCR and in situ hybridization we have shown that prestin is not only expressed in OHCs but is also expressed in vestibular hair cells (VHCs). Immunolocalization studies using peptide specific antibodies confirmed the presence of prestin in VHCs in the mouse, rat and gerbil saccule, utricle, and cristae ampullaris. However, in the VHCs, prestin was not detected in the lateral plasma membrane or in the stereociliary membrane, but was observed in vesicular organelles in the cytoplasm. This suggests that contrary to OHCs, the VHCs do not have an efficient mechanism to target prestin to the plasma membrane. Whole-cell patch-clamp recordings showed that VHCs do not possess the voltage-dependent capacitance associated with electromotility. We conclude that although prestin is expressed in VHCs, it is unlikely that it supports somatic motility like that of OHCs. In a newly initiated project to study the transcriptional repertoire of the organ of Corti during the onset of hearing, a high quality mouse organ of Corti library has been produced and several thousand clones have been sequenced. Preliminary analysis of this EST database showed a large number of potentially novel genes and new splice forms of known genes. Transcripts previously shown to be essential for auditory sensory function as well candidates for deafness are also revealed. One of the most abundant transcripts (0.4%), encoding a novel 89 amino acid protein, was demonstrated to be expressed in inner and outer hair cells by in situ hybridization. The collection of ESTs with description and additional annotations (such as mouse and human chromosomal location) is being compiled as a web-based resource for public access.